Feeder layers comprised of murine thymocytes, fibroblasts, or macrophages have been commonly used to provide growth factors for hybridomas (6, 9). Additional growth factors are especially important during the aminopterin selection for hybridomas and during cloning to ensure monoclonality of the antibodies. However, feeder layers have several drawbacks: their preparation is labor intensive; they are a potential source of contamination; they may utilize nutrients intended for the hybridomas; and they may kill or overgrow the hybridomas they are intended to help. To obviate the later three problems, the hybridoma growth medium may be supplemented with human umbilical cord serum (24), carboxyethyl-gamma-aminobutyric acid (4), bovine hypothalamus extract (17), or the conditioned media of primary cultures of endothelial cells (2) or murine peritoneal macrophages (21).
The preparation of these conditioned media is also a time consuming and laborious task. A technician must obtain cells using sterile technique, count and then culture them at the appropriate density. After a predetermined time the conditioned medium is separated from the cells. One source of the cultured cells, such as macrophages, is the peritoneum of freshly sacrified mice. However, sacrificing and surgery can be distasteful. Additionally, obtaining human specimens is often extremely difficult or impossible, and therefore impractical for routine use.
The use of continuous cell lines, e.g. fibroblasts or macrophages, as a source of hybridoma growth factors has recently been examined. Fazekas De St. Groth and Scheidegger (6) attempted to use continuous macrophage cell lines as a source of feeder layers but found that the feeder layers often overgrew the hybridoma cultures.
It has been shown that the J774A.1, ATCC TIB 67 , P388D1, ATCC TIB63 and RAW264.7, ATCC TIB 71 continuous macrophage cell lines produce a growth factor suitable for hybridoma growth and cloning (15, 22, 23). Mitogen stimulation is necessary for the BJ-1 macrophage cell line to produce the growth factor (3, 22). Conditioned medium prepared from J774A.1 cells without mitogen stimulation was evaluated for hybridoma viability enhancing properties during aminopterin selection but was found to be less effective than a J774A.1 conditioned medium which contained a stimulating mitogen (22, 23). J774A.1 cells which were stimulated for 72 hours with lipopolysaccharide (LPS), washed and then cultured in medium without the added lipopolysaccharide (second harvest) was also evaluated for hybridoma viability enhancing properties. The second harvest conditioned medium was also found to be less effective than the J774A.1 conditioned medium which contained a stimulating mitogen. The enhancement of hybridoma viability was highest in the J774A.1 medium which contained a stimulating mitogen, less in the medium collected from a second harvest in the absence of LPS, and lowest in the conditioned medium harvested from unstimulated J774A.1 cells (23). These data suggest that mitogen-stimulated macrophage cell conditioned media still containing the mitogen yields the desired result of hybridoma viability enhancement during aminoperin selection.
The use of different mitogens to stimulate macrophages to produce proteins such as colony stimulating factor has been well documented (13, 14, 18). Other cells such as B and T lymphocytes can also be stimulated by mitogens to produce lymphokines such as T-cell growth factor or interleukin-2 and osteoclast-activating factor (1, 5, 7, 10, 11). The conditioned medium of a phorbol myristic acetate stimulated thymoma cell line EL-4, serves as a replacement for lymphokines found in the conditioned medium of primary cultures of thymocytes which are necessary for in vitro immunization (12, 19).
The major drawback in using macrophage conditioned media as the source of macrophage-derived hybridoma growth factors (conditioned medium) is that it often contains mitogens which are used to stimulate the production of and/or increase the level of the growth factor in the medium, as well as numerous other enzymes, lymphokines, and cytotoxic factors. The presence of mitogens in the hybridoma growth medium can obscure in vitro immunological studies, e.g. it may stimulate hormone and interferon release or cyclic AMP synthesis (14). A macrophage conditioned medium containing hybridoma growth factors and which has reduced levels of endotoxins or mitogenic substances would be useful for growing hybridomas and minimizing the mitogenic/non-specific effects of various contaminants (e.g. endotoxins, plasminogen activator, interleukin-1, etc.).